Method for preventing and/or treating insulin resistance

ABSTRACT

Described is the use of  Eubacterium hallii  et rel. and/or  Alcaligenes faecalis  et rel., as well as pharmaceutical, food, or feed compositions comprising these bacteria as a medicament, in particular, for preventing and/or treating insulin resistance and/or insulin resistance-related complications such as metabolic syndrome, dyslipidemia and type 2 diabetes mellitus, as well as insulin resistance in endocrine diseases (e.g., obese subjects with type 1 diabetes mellitus, Cushing&#39;s disease and lipodystrophy syndromes. Also described is a method for preventing and/or treating insulin resistance and/or insulin resistance-related complications such as dyslipidemia and type 2 diabetes mellitus as well as insulin resistance in endocrine diseases (e.g., obese subjects with type 1 diabetes mellitus, Cushing&#39;s disease and lipodystrophy syndromes) in a subject in need thereof, the method comprising the step of increasing the level of  Eubacterium hallii  et rel. and/or  Alcaligenes faecalis  et rel. in the small intestine.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase entry under 35 U.S.C. §371 ofInternational Patent Application PCT/NL2012/050592, filed Aug. 30, 2012,designating the United States of America and published in English asInternational Patent Publication WO 2013/032328 A1 on Mar. 7, 2013,which claims the benefit under Article 8 of the Patent CooperationTreaty and under 35 U.S.C. §119(e) to U.S. Provisional PatentApplication Ser. No. 61/528,931, filed Aug. 30, 2011, and to TheNetherlands Patent Application Serial No. 2007319, filed Aug. 30, 2011.

TECHNICAL FIELD

The disclosure relates to the field of medicine. This disclosure isdirected to bacteria of the taxa Eubacterium hallii et rel. and/orAlcaligenes faecalis et rel., optionally used in a pharmaceutical, food,or feed composition, for use as a medicament, in particular, forpreventing and/or treating insulin resistance and/or insulinresistance-related complications such as metabolic syndrome,dyslipidemia and type 2 diabetes mellitus.

BACKGROUND

Obesity is primarily a consequence of detrimental nutritional andphysical habits against an unfavorable genetic background. It is a majorrisk factor for the development of common medical conditions such as themetabolic syndrome, type 2 diabetes mellitus and cardiovascular disease.As a metabolically active organ, the human intestine contains a denseand diverse community of micro-organisms, dominated by over a thousanddifferent bacterial species. There is growing evidence for the role ofintestinal microbiota in host metabolism.

The phyla that account for the vast majority of intestinal microbiotainclude the Gram-negative Bacteroidetes, Proteobacteria andVerrucomicrobia, as well as the Gram-positive Firmicutes andActinobacteria. It was previously shown that the gut microbiotacontributes to the development of diet-induced obesity in mice. Thecolonic microbiota in obese mice appeared to be characterized by a lowermicrobial diversity and an enrichment in carbohydrate andlipid-utilizers. Putatively, the short chain fatty acids acetate,propionate and butyrate produced by specific gut bacteria could serve asa signal that directly influences host hepatic and peripheral insulinsensitivity. On the other hand, recent research showed that lower gutmicrobial diversity in mice was associated with endotoxemia-inducedchronic inflammation and subsequent development of insulin resistance.

In humans, altered colonic microbiota have been correlated to obesity,but consensus regarding specific bacterial groups of species andevidence for a causative role is lacking. As metabolically healthy andunhealthy obese phenotypes exist based on the absence or presence ofinsulin resistance, published reports on associations between intestinalmicrobiota composition and human obesity seem to be compromised byheterogeneity in the obese phenotype, various confounding factors e.g.,diet, medication use) and developing methods to analyze the intestinalmicrobiota. This is particularly true for the small intestinalmicrobiota that is relatively inaccessible but is exposed to a largesurface. It has been found that the microbial diversity of the smallintestine is smaller than that of the colon and is notably enriched inbacteria belonging to the Lactobacillales and Veillonella spp. (Booijnket al., 2010, Env. Microbiol. 12:3213-27). Thus, there is a need in theart to find further medicaments suitable to treat and/or prevent insulinresistance and/or type 2 diabetes mellitus, preferably medicaments thatcan be easily incorporated in the patient's lifestyle, for example, inthe form of food compositions for daily consumption.

DISCLOSURE

The disclosure relates to Eubacterium hallii et rel. and/or Alcaligenesfaecalis et rel. for use in preventing and/or treating insulinresistance and/or insulin resistance-related conditions like metabolicsyndrome, dyslipidemia and type 2 diabetes mellitus, as well as insulinresistance in endocrine diseases (e.g., obese subjects with type 1diabetes mellitus, Cushing's disease and lipodystrophy syndromes).

In a second aspect, provided is a pharmaceutical, food, or feedcomposition comprising Eubacterium hallii et rel. and/or Alcaligenesfaecalis et rel. for use in preventing and/or treating insulinresistance and/or related conditions like metabolic syndrome,dyslipidemia and type 2 diabetes mellitus, as well as insulin resistancein endocrine diseases (e.g., obese subjects with type 1 diabetesmellitus, Cushing's disease and lipodystrophy syndromes).

In a further aspect, this disclosure pertains to a method for preventingand/or treating insulin resistance and/or related conditions in asubject in need thereof, the method comprising the step of increasingthe level of Eubacterium hallii et rel. and/or Alcaligenes faecalis etrel. in the small intestine. The level of Eubacterium hallii et rel.and/or Alcaligenes faecalis et rel. in the small intestine may beincreased by a method selected from the group consisting ofadministering an effective amount of Eubacterium hallii et rel. and/orAlcaligenes faecalis et rel. to the subject, and administering aneffective amount of a compound capable of increasing the level ofEubacterium hallii et rel. and/or Alcaligenes faecalis et rel. in thesmall intestine.

In another aspect, the disclosure pertains to a pharmaceutical, food, orfeed composition comprising Alcaligenes faecalis et rel. The compositionmay be a drink. The pharmaceutical, food, or feed composition comprisingAlcaligenes faecalis et rel. may be for use as a medicament.

Definitions

As used herein, the term “insulin resistance” has its common meaning inthe art. Insulin resistance is a physiological condition where thenatural hormone insulin becomes less effective at lowering blood sugars.The resulting increase in blood glucose may raise levels outside thenormal range and cause adverse health effects such as metabolicsyndrome, dyslipidemia and subsequently type 2 diabetes mellitus. Theterm “insulin resistance-related complications” and “insulinresistance-related conditions” as used herein encompass, withoutlimitation, metabolic syndrome, dyslipidemia and type 2 diabetesmellitus, as well as insulin resistance in endocrine diseases (e.g.,obese subjects with type 1 diabetes mellitus, Cushing's disease andlipodystrophy syndromes).

The addition “et rel.” behind the genus-like group name (level 2 groupname) stands for “et relatives,” indicating all relatives of thisphylogenetic group, i.e., those indicated in Table 3 of WO 2011/043654(which is herein incorporated by reference), in the column headed “level3.” This information, including the indicated 16S rRNA gene sequences,can be used to develop specific PCR primers or LCR probes to detect theone or more members of these groups. In some literature, the addition“et rel.” is replaced by “-like” to indicate the fact that the groupincludes more than one related species. However, this is a ratherambiguous designation and hence all terms with “et rel.” are clearlydefined in Table 3 of the incorporated WO 2011/043654, which has alsobeen published by Rajilic-Stojaniovic et al. 2007, Environ. Microbiol.9(9):2125-2136).

In the context of the disclosure, a subject may be an animal or a humanbeing. Preferably, the subject is a human being. A “healthy subject,” asreferred to herein, does not suffer from insulin resistance and/ordiabetes mellitus, and, preferably, does not suffer from any conditionsor diseases of the gastrointestinal tract, and, more preferably, doesnot suffer from any known conditions or diseases. Preferably, a “healthysubject,” as referred to herein, has a Body Mass Index (BMI) in therange of between 18.5 and 24.9 kg/m².

As used herein, the level of bacteria of the taxa Eubacterium hallii etrel. and/or Alcaligenes faecalis et rel. in a sample, e.g., anintestinal sample (e.g., duodenal or fecal), is increased when it issignificantly higher than the level of one or more bacteria in a controlsample, e.g., an intestinal control sample (e.g., duodenal or fecal). Itis also considered increased when the level of bacteria of the taxaEubacterium hallii et rel. and/or Alcaligenes faecalis et rel. in asample is at least 5%, such as 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50% higher than the bacteria of the taxa Eubacterium hallii et rel.and/or Alcaligenes faecalis et rel. in the control sample. The “controlsample,” as used herein, refers to a sample taken from a subjectreceiving treatment by administration of bacteria of the taxaEubacterium hallii et rel. and/or Alcaligenes faecalis et rel. prior toadministration of bacteria of the taxa Eubacterium hallii et rel. and/orAlcaligenes faecalis et rel., optionally in an effective amount.

As used herein, the term “effective amount” refers to a quantitysufficient to achieve a desired therapeutic and/or prophylactic effect,e.g., an amount that results in the treatment and/or prevention ofinsulin resistance and/or related complications like dyslipidemia andtype 2 diabetes mellitus as well as insulin resistance in endocrinediseases (e.g., obese subjects with type 1 diabetes mellitus, Cushing'sdisease and lipodystrophy syndromes). In the context of therapeutic orprophylactic applications, the amount of bacteria administered to thesubject will depend on the type and severity of the disease or conditionand on the characteristics of the subject, such as general health, age,sex, body weight and tolerance to drugs. It will also depend on thedegree, severity and type of disease or condition. The skilled artisanwill be able to determine appropriate dosages depending on these andother factors. The bacteria can also be administered in combination withone or more additional therapeutic compounds. For example, with thephrase a “therapeutically effective amount” of the bacteria is meantlevels of the bacteria that lead to an improvement of the physiologicaleffects of a disease or condition associated with insulin resistanceand/or related complications like dyslipidemia and type 2 diabetesmellitus, as well as insulin resistance in endocrine diseases (e.g.,obese subjects with type 1 diabetes mellitus, Cushing's disease andlipodystrophy syndromes). The skilled person will be capable ofdetermining when such disease or condition has been treated and/orprevented.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. In addition, the verb “to consist” may be replaced by“to consist essentially of,” meaning that a composition of thisdisclosure may comprise additional component(s) than the onesspecifically identified, those additional component(s) not altering theunique characteristics of the disclosure.

In addition, reference to an element by the indefinite article “a” or“an” does not exclude the possibility that more than one of the elementis present, unless the context clearly requires that there be one andonly one of the elements. The indefinite article “a” or “an” thususually means “at least one.”

DETAILED DESCRIPTION

We have found a causal role of the small intestinal microbiota ininsulin resistance and dyslipidemia. Eighteen male subjects with newlydiagnosed metabolic syndrome underwent small intestine biopsies andsubsequent polyethylene-glycol bowel lavage through duodenal tubeinsertion followed by random assignment to either allogenic orautologous fecal transplantation. In the allogenic fecal transplantationgroup that was performed on nine subjects, the fecal material wasderived from a healthy and lean donor. The autologous transplantationgroup included the nine other subjects and these received their ownfecal material.

It was found that the subjects of the allogenic group were characterizedby different sigmoidal gut microbiota compared to those of theautologous group as determined by analysis with a phylogeneticmicroarray (the Human Intestinal Tract Chip, HITChip)(Rajilic-Stojanovic, 2009, Environ. Microbiol. 11(7):1736-1751). Fastinglevels of TG-rich lipoproteins (TG/ApoB ratio) were significantlyreduced in the subjects in the allogenic group with no effect afterautologous feces infusion. Although the weight of the subjects remainedstable, six weeks after feces transplantation, an improvement in bothperipheral (Rd) and hepatic insulin sensitivity (suppression of EGP) wasfound six weeks in the allogenic group while no significant changes wereobserved in the autologous treatment group.

We have identified changes in small intestinal microbiota betweensubjects receiving allogenic or autologous fecal transplantation.Comparing the small intestinal microbiota composition at baseline andafter six weeks in the allogenic group showed an increased abundance ofbacteria related to the ileum-inhabitant Alcaligenes faecalis and thebutyrate-producing Eubacterium hallii. Notably, the latterbutyrate-producer was almost two-fold reduced following infusion in theautologous group. Bacteria belonging to Eubacterium hallii et rel.include relatively fast-growing anaerobes. They have the metaboliccapacity to convert lactate into butyrate in a process that needsacetate (Munoz-Tamayo et al., 2011, FEMS Microbiol. Ecolo. 76:615-624).Lactate and acetate are abundant metabolites in the upper intestinaltract that is colonized by, among others, streptococci and lactobacillithat can produce these compounds (Booijink et al., 2010, vide supra).However, it may be a specific embodiment of the disclosure to includethe substrates lactate and acetate to the formulation containingbacteria belonging to the taxon Eubacterium hallii et rel. Bacteriarelated to Alcaligenes faecalis (belonging to the taxon Alcaligenesfaecalis et rel.) are facultative anaerobic bacteria that degrade avariety of substrates—they have the unusual capacity to produce nitrousand nitric oxide under low oxygen conditions in the presence of ammonia(Anderson et al., 1993, Appl. Environ. Microbiol. 95:3525-33). As theseconditions are met in the upper intestine, it is feasible thatAlacaligenes faecalis produces nitric oxide. It has been proposed thatnitric oxide is a therapy for the treatment of patients with type 2diabetes and metabolic syndrome (Ahanchi et al., 2008, Am. J. Physiol.Heart Circ. Physiol. 295:H2388-98). However, delivery of nitrous oxidevia its production by intestinal bacteria has not been described.

Thus, the disclosure relates to bacteria of the taxon Eubacterium halliiet rel. and/or bacteria of the taxon Alcaligenes faecalis et rel. foruse in preventing and/or treating insulin resistance and/or insulinresistance-related complications such as metabolic syndrome,dyslipidemia and type 2 diabetes mellitus as well as insulin resistancein endocrine diseases (e.g., obese subjects with type 1 diabetesmellitus, Cushing's disease or lipodystrophy syndromes. In anotherembodiment, the present disclosure relates to bacteria of the taxonEubacterium hallii et rel. and/or bacteria of the taxon Alcaligenesfaecalis et rel. for use in preventing and/or treating a clinicalcondition in a mammal, such as human, which results from the endogenoushormone insulin becoming less effective at lowering blood sugars andsubsequent plasma cholesterol profiles. Non-limiting examples of suchclinical conditions include metabolic syndrome, dyslipidemia and type 2diabetes mellitus as well as insulin resistance in endocrine diseases(e.g., obese subjects with type 1 diabetes mellitus, Cushing's diseaseand lipodystrophy syndromes). Bacteria of either of the taxa may be usedalone as a medicament for the indicated purposes, or bacteria of thetaxon Eubacterium hallii et rel. and bacteria of the taxon Alcaligenesfaecalis et rel. may be used together as a medicament. Moreover, acombination of any one of these taxa of bacteria or bacteria of eithertaxa together may be used with currently used therapeutic agents inclinical practice (e.g., biguanides, sulfonureum derivates, PPAR gammaagonists, DPPIV inhibitors and injectable medication like GLP1 agonistand/or exogenous short-/long-acting insulin).

The disclosure also relates to a pharmaceutical, food, or feedcomposition comprising Eubacterium hallii et rel. and/or Alcaligenesfaecalis et rel. for use in preventing and/or treating insulinresistance and/or related complications like dyslipidemia and type 2diabetes mellitus. The pharmaceutical, food or feed compositionpreferably comprises an effective amount of Eubacterium hallii et rel.and/or Alcaligenes faecalis et rel. Preferably, the pharmaceutical, foodor feed composition comprises in total between about 10⁶ and about 10¹²,preferably between about 10⁸ and about 10¹², bacteria of the taxonEubacterium hallii et rel. and/or bacteria of the taxon Alcaligenesfaecalis et rel. Preferably, the bacteria are contained in a daily dose.

In another aspect, the disclosure pertains to a pharmaceutical, food, orfeed composition comprising Alcaligenes faecalis et rel., optionally foruse as a medicament. Such composition may comprise a carrier, such as aninert carrier.

Preferably, the composition referred to herein is for enteral or oraladministration. A composition for enteral or oral administration may beeither a food composition, feed composition, or a pharmaceuticalcomposition. Such food composition, feed composition, or pharmaceuticalcomposition does not include fecal compositions or compositions derivedfrom fecal compositions.

A pharmaceutical composition will usually comprise a carrier, such as apharmaceutical carrier, in addition to bacteria of the taxon Eubacteriumhallii et rel. and/or bacteria of the taxon Alcaligenes faecalis et rel.The carrier is preferably an inert carrier. The preferred form dependson the intended mode of administration and (therapeutic) application. Apharmaceutical carrier can be any compatible, nontoxic substancesuitable to deliver bacteria of the taxon Eubacterium hallii et rel.and/or bacteria of the taxon Alcaligenes faecalis et rel. to thegastro-intestinal tract of a subject. For example, sterile water, orinert solids may be used as a carrier usually complemented with apharmaceutically acceptable adjuvant, buffering agent, dispersing agent,and the like. A composition will either be in liquid, e.g., a stabilizedsuspension of bacteria of the taxon Eubacterium hallii et rel. and/orbacteria of the taxon Alcaligenes faecalis et rel., or in solid forms,e.g., a powder of lyophilized bacteria of the taxon Eubacterium halliiet rel. and/or bacteria of the taxon Alcaligenes faecalis et rel. Incase of lyophilization, a cryoprotectant such as lactose, threhalose orglycogen can be envisaged. For example, for oral administration,bacteria of the taxon Eubacterium hallii et rel. and/or bacteria of thetaxon Alcaligenes faecalis et rel. can be administered in solid dosageforms, such as capsules, tablets, and powders, or in liquid dosageforms, such as elixirs, syrups, and suspensions. Bacteria of the taxonEubacterium hallii et rel. and/or bacteria of the taxon Alcaligenesfaecalis et rel. can be encapsulated in capsules such as gelatincapsules, together with inactive ingredients and powdered carriers, suchas, e.g., glucose, lactose, sucrose, mannitol, starch, cellulose orcellulose derivatives, magnesium stearate, stearic acid, sodiumsaccharin, talcum, magnesium carbonate and the like.

A preferred composition according to the disclosure is suitable forconsumption by a subject, which is preferably a human or a non-humananimal. Such compositions may be in the form of a food supplement or afood or food composition (herein jointly referred to as “foodcomposition”), which, besides bacteria of the taxon Eubacterium halliiet rel. and/or bacteria of the taxon Alcaligenes faecalis et rel., alsocontains a suitable food base. Alternatively, such composition may be inthe form of a feed supplement or a fodder or feed composition (hereinjointly referred to as “feed composition”). A food or food compositionor feed composition is herein understood to include a liquid for humanor non-human animal consumption, i.e., a drink or beverage. A food orfood composition or feed composition may be a solid, semi-solid and/orliquid food or food composition and, in particular, may be a dairyproduct, such as a fermented dairy product, including, but not limitedto a yogurt, a yogurt-based drink or buttermilk. Such a food or foodcomposition or feed composition may be prepared in a manner known perse, for example, by adding bacteria of the taxon Eubacterium hallii etrel. and/or bacteria of the taxon Alcaligenes faecalis et rel. to asuitable food, food base, or feed base, in a suitable amount. Similarly,this may include the use of these bacteria in capsulated form asdescribed above since they have to pass the low pH of the stomach. Thismay also be a preferred way so as to reduce the traces of butyrate thatare associated with the growth of bacteria belonging to the taxonEubacterium hallii et rel. and may produce off-flavor in a food or foodcomposition. In another embodiment, bacteria of the taxon Eubacteriumhallii et rel. and/or bacteria of the taxon Alcaligenes faecalis et rel.may be used in or for the preparation of a food or food composition orfeed composition, e.g., by fermentation. In doing so, bacteria of thetaxon Eubacterium hallii et rel. and/or bacteria of the taxonAlcaligenes faecalis et rel. may be used in a manner known per se forthe preparation of such fermented foods or food compositions orfermented feed compositions, e.g., in a manner known per se for thepreparation of fermented dairy products using lactic acid bacteria. Insuch methods, bacteria of the taxon Eubacterium hallii et rel. and/orbacteria of the taxon Alcaligenes faecalis et rel. may be used inaddition to a micro-organism usually used, and/or may replace one ormore or part of a micro-organism usually used.

Preferably, the above compositions will contain bacteria of the taxonEubacterium hallii et rel. and/or bacteria of the taxon Alcaligenesfaecalis et rel. in amounts that allow for convenient (oral)administration as indicated above, e.g., as or in one or more doses perday or per week. In particular, a preparation may contain a unit dose ofbacteria of the taxon Eubacterium hallii et rel. and/or bacteria of thetaxon Alcaligenes faecalis et rel.

In a further aspect, the disclosure relates to a method for preventingand/or treating insulin resistance and/or related complications likedyslipidemia and type 2 diabetes mellitus in a subject in need thereof,the method comprising the step of increasing the level of Eubacteriumhallii et rel. and/or Alcaligenes faecalis et rel. in the smallintestine.

The level of bacteria of the taxon Eubacterium hallii et rel. and/orbacteria of the taxon Alcaligenes faecalis et rel. may be measured bydetermining the levels of nucleic acid sequences, amino acid sequencesand/or metabolites specific for one or more bacteria, preferably thelevel of nucleic acid sequences specific for one or more bacteria.

The level of one or more bacteria may preferably be measured bydetermining the level of specific nucleic acid sequences in a testsample derived from the small intestine, which nucleic acid sequencesare preferably 16S rRNA gene sequences of bacteria of the taxonEubacterium hallii et rel. and/or bacteria of the taxon Alcaligenesfaecalis et rel., more preferably, one or more variable regions of the16S rRNA gene sequences, e.g., one or more of the variable regions V1and/or V6 of the 16S rRNA gene sequences.

The level of Eubacterium hallii et rel. and/or Alcaligenes faecalis etrel. in the small intestine may be increased by a method selected fromthe group consisting of administering an effective amount of Eubacteriumhallii et rel. and/or Alcaligenes faecalis et rel. to the subject, andadministering an effective amount of a compound capable of increasingthe level of Eubacterium hallii et rel. and/or Alcaligenes faecalis etrel. in the small intestine.

Compounds capable of increasing the level of Eubacterium hallii et rel.in the small intestine may include, without limitation, lactate andacetate. Alternatively, Eubacterium hallii et rel. may be administeredin combination with lactic acid-producing bacteria such as Lactobacillusspp. and Bifidobacterium spp. The lactic acid producing bacteria may bepresent in a fermented food product such as yogurt or a yogurt drink perse, and Eubacterium hallii et rel. may be added. Compounds capable ofincreasing the level of Alcaligenes faecalis et rel. in the smallintestine may include, without limitation, substrates allowingproduction of nitrous and nitric oxide under low oxygen conditions inthe presence of ammonia.

In an embodiment, the bacteria of the taxon Eubacterium hallii et rel.are bacteria from the Eubacterium hallii strain L2-7. The Eubacteriumhallii strain L2-7 (DSM 17630) is available from the Deutsche Sammlungvon Mikroorganismen (DSMZ). Bacteria of the taxon Alcaligenes faecaliset rel. may, for example, be cultured in accordance with Annamalai etal. (Ann. Microbiol. December, 2011, 61(4):801-807).

The skilled person will be capable of selecting an effective amount of acompound capable of increasing the level of Eubacterium hallii et rel.and/or Alcaligenes faecalis et rel. in the small intestine using methodsthat are routine in the art.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

It will be clear that the above description is included to illustratesome embodiments of the disclosure, and not to limit the scope ofprotection. Starting from this disclosure, many more embodiments thatare within the scope of protection and the essence of this disclosureand that are obvious combinations of prior art techniques and thedisclosure hereof will be evident to a skilled person.

EXAMPLES Example 1 Methods

A double-blind randomized controlled trial was conducted in which theeffect of a single allogenic (lean donor) microbial fecal infusion onglucose metabolism in relation to gut microbiota composition wasinvestigated in obese subjects.

Subjects

Male Caucasian obese subjects were screened for characteristics of themetabolic syndrome comprising waist circumference>102 cm and fastingplasma glucose>5.6 mmol/1.17. Subjects with cholecystectomy and/or usingany medication, probiotics and/or antibiotics in the past three monthswere excluded. Written informed consent was obtained from all subjects.The study was approved by the Institutional Review Board and conductedin accordance with the principles of the Declaration of Helsinki (1996).The study was registered at the online Dutch Trial Register (NTR1776).

Screening of Lean Donors

Lean Caucasian males (BMI<23 kg/m²) were also recruited by newspaperadvertisements. They completed a questionnaire regarding bowel habits,travel history, comorbidity and medication use. They were screened forthe presence of infectious diseases according to an adapted version ofthe questionnaire of the Dutch Blood Transfusion service (Sanquin)(Langeveld et al., 2008, J. Clin. Endocrinol. Metab. 93(3):845-851).Blood was screened for the presence of antibodies to humanimmunodeficiency virus; human T-lymphotropic virus; Hepatitis A, B, andC; cytomegalovirus; Epstein-Barr virus; Strongyloides; and amoebiasis.Donors were also excluded if screening of their feces revealed thepresence of parasites (e.g., Blastocystis hominis or Dietamoebafragilis), Clostridium difficile and possible other pathogenic bacteria(Shigella, Campylobacter, Yersinia, Salmonella).

Experimental Design

Glucose metabolism was measured in the basal state and during a two-stephyperinsulinemic euglycemic clamp to measure endogenous glucoseproduction (EGP), hepatic and peripheral insulin sensitivity (Rate ofdisposal, Rd) using [6,6 2H2]-glucose. Body weight was recorded and bodycomposition was measured using bioimpedance analysis. Resting energyexpenditure (REE) and respiratory quotient were measured using indirectcalorimetry (Langeveld, J. Clin. Endocrinol. Metab. 2008,93(3):845-851).

Participants were allowed to keep their own diet, but were asked to keepa weekly online nutritional diary (www.dieetinzicht.nl) to monitorcaloric intake. After an overnight fast, study subjects and donorsbrought freshly produced morning stool for processing; study subjectswere randomized in a double-blind fashion to either allogenic (from leanmale donors with BMI<23 kg/m²) or autologic (own collected feces) gutmicrobial infusion via gastro-duodenal infusion (see procedure). Studysubjects first underwent gastroduodenoscopy and small intestinal(jejunal) biopsies were taken near Treitz ligament. Biopsy samples werecollected in sterile tubes, snap-frozen in liquid nitrogen and processedas described earlier (Langeveld et al., supra). A duodenal tube waspositioned and bowel lavage with macrogol solution was performed overfive hours to clean out endogenous fecal contamination followed by gutmicrobial infusion. Gastroduodenoscopy-assisted biopsies and thehyperinsulinemic euglycemic clamp were repeated six weeks aftertransplantation.

Hyperinsulinemic Euglycemic Clamp

After a twelve-hour fast, a catheter was inserted into an antecubitalvein for infusion of stable-isotope tracer [6,6-2H2]glucose (CambridgeIsotopes, Andover, Mass.), insulin and glucose. A second catheter wasinserted retrogradely in the contralateral hand vein and kept in athermo-regulated (60° C.) clear plastic box for sampling of arterializedvenous blood. Saline was infused as 0.9% NaCl at a rate of 50 mL/hour tokeep the catheters patent. At t=0 hour (0800), blood samples were drawnfor determination of background enrichments. Then, a primed continuousinfusion of isotopes was started: [6,6-2H2]glucose (prime: 8.8 μmol/kg;continuous: 0.11 μmol·kg-1·min-1) and continued until the end of theclamp. After a two-hour equilibration period, blood samples were drawnfor isotope enrichments and samples for gluco-regulatory hormones, freefatty acids (FFAs) and incretins. Thereafter (t=2.0 hours), a two-stephyperinsulinemic euglycemic clamp was started: step 1 included aninfusion of insulin at a rate of 20 mU·m-2·min-1 (ACTRAPID™ 200 IU/mL;Novo Nordisk Farma BV, Alphen aan den Rijn, Netherlands) to assesshepatic insulin sensitivity. Glucose 20% was started to maintain aplasma glucose concentration of 5 mmol/L. Plasma glucose concentrationswere measured every five minutes at the bedside using a Beckman glucosemeter. After two hours (t=4 hours), blood samples were drawn atfive-minute intervals for the measurement of glucose concentrations andisotopic enrichments. Another blood sample was drawn for measurement ofgluco-regulatory hormones and FFAs. Hereafter, insulin infusion wasincreased to a rate of 60 mU·m-2·min-1 (step 2) to assess peripheralinsulin sensitivity. After another two hours (t=6 hours), blood samplingwas repeated.

Body composition was measured at baseline and after six weeks withbioelectrical impedance analysis (Maltron BF906; Maltron, Rayleigh, UK).Oxygen consumption (VO₂) and CO₂ production (VCO₂) were measuredcontinuously during the final 20 minutes of both the basal state and thehyperinsulinemic euglycemic clamp by indirect calorimetry using aventilated hood system (Sensormedics model 2900; Sensormedics, Anaheim,Calif.). REE, carbohydrate oxidation (CHO), and fatty acid oxidation(FAO) rates were calculated from oxygen consumption and carbon dioxideproduction. Rate of appearance (Ra) and rate of disappearance (Rd) ofglucose were calculated using the modified form of the Steele equationsfor non-steady-state measurements as described previously. Endogenousglucose production (EGP) was calculated as the difference between Raglucose and glucose infusion rate. Both peripheral (Rd) and hepaticinsulin sensitivity (suppression of EGP) were calculated and expressedas median with range.

Gut Microbiota Analysis DNA Isolation

DNA was isolated and purified using the repeated bead-beating pluscolumn method as described previously (Zoetendal, Syst. Appl. Microbiol.24(3):405-410). For DNA isolation of the biopsies, we used a differentbead-beating protocol (Nadkarni et al., 2002, Microbiology 148(Pt1):257-266). In short, 0.5 gram (wet weight) of feces was suspended inLysis buffer (500 mM NaCl, 50 mM Tris-HCl pH 8, 50 mM EDTA, 4% SDS) plusZirconia beads and glass beads. The tube was shaken with FASTPREP® (atsetting 5.5) for three minutes at 4° C., followed by incubation at 95°C. for 15 minutes. The DNA in the supernatant was precipitated withammonium acetate and isopropanol, washed with 70% ethanol and afterwardtreated with proteinase K and DNase-free RNase. Finally, the DNA waspurified on a QIAAMP® spin column (Qiagen) according to themanufacturer's instructions. DNA concentration was quantified using theNANODROP® 1000 spectrophotometer (NanoDrop Technologies, Wilmington,Del.).

HITChip Microbiota Profiling

The HITChip was used for phylogenetic profiling of the microbiota infeces and small intestinal biopsies as described previously(Rajilic-Stovanojic, 2009, supra). In short, 10 ng DNA was used toamplify the 16S rRNA genes using the T7prom-Bact-27-for and Uni-1492-revprimers followed by in vitro transcription and labeling with Cy3 andCy5, respectively, for fecal samples. The primer Prok-1369-rev was usedas reverse primer for the biopsy samples because Uni-1492-rev wasmajorly targeting the overabundant human DNA, resulting in its depletionfor efficient bacterial 16S rRNA gene amplification (data not shown).Equimolar mixes of Cy3/Cy5-labeled 16S rRNA targets were fragmented andsubsequently hybridized on the microarrays at 62.5° C. for 16 hours in arotation oven (Agilent Technologies, Amstelveen, The Netherlands),followed by washing and drying of the slides. Samples were arrayed induplex (technical replication). After scanning of the slides, the datawas extracted from the microarray images using the Agilent FeatureExtraction software, versions 7.5-9.1 (on their website at agilent.com).Subsequently, the microarray data were min-max normalized and furtheranalyzed using a set of R-based scripts (see website at r-project.org/)in combination with a custom designed relational database that runsunder the MySQL database management system (see website at mysql.com).Hierarchical clustering of probe profiles was carried out usingcorrelation-based distance and complete linkage method.

Fecal Transplant Procedure

The patient and donor delivered freshly produced stool at the day ofinfusion (approximately 200 grams, produced within six hours beforeuse). Fecal samples (either allogenic or autologous) were taken beforeand after processing to study procedural effects on microbialcomposition. After delivery, the feces was covered with 500 cc sterilesaline (0.9% NaCl), transferred to a blender and mixed for ten minutes.The homogenized solution was then filtered twice through a clean metalsieve. Subsequently, the filtrate was transferred to a 1000 ml sterileglass bottle and stored at room temperature until the patient hadfinished the bowel lavage. Finally, the fecal microbial solution wasgradually infused through the duodenal tube, in approximately 30minutes.

Biochemistry

Fasting plasma samples were obtained for measurement of totalcholesterol, LDL cholesterol (LDLc), HDL cholesterol (HDLc) andtriglycerides (TG), using commercially available enzymatic assays(Randox, USA and Daiichi, Japan). All analyses were performed using aCobas Mira autoanalyzer (Horiba, France). LPS-binding Protein (LBP) andC-Reactive Protein (CRP) were measured using commercial ELISA (HyCult,USA and Roche, Switzerland). Fecal short-chain fatty acid concentrationscomprising acetate, butyrate and propionate were analyzed as previouslydescribed (Wolever et al., 2000, Br. J. Nutr. 84(1):57-61).

Intestinal Microbiota and Host Mucosa Response Analyses

A morning stool sample collected at baseline and after six weeks,respectively, was obtained from donor and study subjects to determinethe microbiota composition. Samples were collected into two plasticcontainers, immediately frozen at −20° C. and transferred to −80° C.within a week. The microbiota composition of the small intestinalbiopsies and fecal samples was determined by using the Human IntestinalTract Chip (HITChip), a custom-made Agilent microarray (AgilentTechnologies, Palo Alto, Calif., USA) containing approximately 5,500oligonucleotide probes that cover over 1,000 intestinal phylotypes(Rajilic-Stojanovic et al. 2009, vide supra). Quantification of totalbacteria and methanogens was performed by 16S rRNA gene quantitative PCRwith the same DNA used for HITChip analysis. Small intestinal biopsytranscriptome raw data using Human HT-12 v3 expression arrays (Illumina,San Diego, USA) were uploaded on Gene Expression Omnibus (registrationnumber: GSE30854). Details of both intestinal microbiota and arrayanalysis are provided in the Supplementary Appendix.

Statistical Analysis

Statistical analyses were performed with SPSS software, version 16. Dataare expressed as means±standard error of mean (normal distribution) ormean (skewed distribution). To compare data between groups, Student'st-test (normal distribution) or Wilcoxon Signed rank test (skeweddistribution) was used. All reported P values are two-sided. Expressionanalysis for the HITChip and Illumina arrays was carried out with linearmixed and random forest methods as well as canonical correlation (CCA)analysis. Statistical tests were performed using Microsoft Office EXCEL®or R statistical software (http://r-project.org/).

Statistical Analysis of HITChip and Illumina Array

Expression analysis for HITChip and Illumina array was carried out withNLME package (Pinheiro and Bates, Mixed-effect models in S and S-plus,Springer; 2000). A linear mixed model with effects for time (0 or 6weeks), treatment (autologous or allogenic), and a cross effect of thetwo main effects was constructed. Repeated measures design of theexperiment was taken into account by including a patient-specific randomeffect. For each measurement unit (gene or bacteria), contrasts werecomputed using the multcomp package, and the p-values thus obtained weresubjected to correction for multiple comparisons by q-value package(Bretz, HTWP, Multiple Comparisons Using R, CRC Press, Boca Raton, 2010;Storey J. A., A direct approach to false discovery rates, Journal of theRoyal Statistical Society Series B (statistical methodology) 2010,64(3):479-498). Individual temporal stability of the fecal microbiota inthe patients of both groups was determined by computing a Pearsoncorrelation in oligonucleotide level between the samples taken at thetime of the transplantation and those obtained after six weeks.

In jejunal samples, bacterial groups associated with the differencebetween the allogenic and autologous groups were determined with theRandom Forest method using the bacterial composition changes before andsix weeks after transplantation as covariates. Bootstrap averaging(bagging) (Breiman, Bagging Predictors, Machine Learning 1996, 24(2))combined with redundancy analysis was then used to get a robust estimateof the groups contributing to the difference, to estimate the p-value ofthe separation, and to visualize the result. Association between geneexpression and jejunal samples was determined using sparse canonicalcorrelation analysis (sparse CCA). To reduce the effect of overfitting,the set of genes to be correlated consisted of top ten differentiallyexpressed genes. The microbiota data consisted of HITCHip data on sixtaxa found to be significantly contributing to the difference betweenautologous and allogenic samples in the jejunal samples. In CCAanalysis, regularization parameters were first estimated withleave-one-out cross-validation. Then the model was repeated with alldata, and for each variable, the correlations to the canonical variateswere computed.

Results Baseline Characteristics

A total of 44 male obese subjects were screened for features of themetabolic syndrome and 20 eligible subjects were included. Two subjectswere excluded from analyses due to antibiotic use during the trialunrelated to the microbial transplant. Therefore, eighteen subjects wereavailable for analysis.

Effect of Fecal Transplant on Insulin Sensitivity, Fecal SCFA and LBP

Seven healthy lean donors, one of which provided multiple donations,were used for the allogenic transplantation of nine obese subjects withthe metabolic syndrome. Equal amounts of feces were infused in the obesesubjects from either allogenic or autologous microbial fecal infusion(190±33 and 187±47 gram, ns). Moreover, the processing time betweenfeces production and infusion did not differ (5.8±0.8 and 6.1±1.2 hoursin the allogenic and autologous groups, respectively). None of the obesesubjects experienced any adverse events during the trial or developedIrritable Bowel Syndrome symptoms according to the Rome III criteria.

Body weight remained stable in both groups between baseline and sixweeks (allogenic: from 122.7±19 to 122.5±19 kg versus autologous:113.2±20 to 113.4±20 kg, ns). No effect on daily caloric dietary intake,resting energy expenditure or carbohydrate/fatty acid oxidation was seenin both groups after microbial fecal infusion (data not shown). Therewas a marked improvement in peripheral insulin sensitivity six weeksafter allogenic feces treatment (median Rd: from 26.2 to 45.3μmol/kg/minute, p<0.05), while no significant change was observed in theautologous treatment group (median Rd: from 21.0 to 19.5 μmol/kg/minute,ns). A trend toward improvement in hepatic insulin sensitivity,expressed as EGP suppression from basal was observed (median EGPsuppression: from 51.5 to 61.6%, p=0.08), while no effect was observedin the autologous treatment group (median EGP suppression: from 53.8 to52.4%, ns). There were no changes in gluco-regulatory hormones, eitherin the basal state or during hyperinsulinemia (data on file) in bothgroups.

Lean donors were characterized by an increased fecal harvest of butyrateand propionate compared to obese participants, a trait that was alsoobserved upon allogenic microbial fecal infusion. Moreover, we found asignificant decrease of lipopolysaccharide-binding protein (LBP) sixweeks after lean donor transplant (median LBP: from 19.9 to 18.6 μg/ml(p<0.05 and median CRP from 1.5 to 1.6 mg/L, ns) with no significantchanges in the autologous group (median LBP: from 23.0 to 22.3 μg/ml andmedian CRP from 3.1 to 2.5 mg/L, ns).

Effect of Fecal Transplant on Gut Microbiota in Feces

The fecal microbiota of the obese subjects were characterized by lowergut microbial diversity, higher amounts of Bacteroidetes and decreasedamounts of Clostridium cluster XIVa bacteria as compared to lean donorsubjects (data not shown). To determine the impact of the microbialtransplantation, we compared the fecal microbiota at baseline and aftersix weeks. Total numbers of fecal bacteria did not change followingmicrobial fecal infusion. At six weeks, the analysis on the genus-likelevel showed a clear separation of the samples belonging to allogenicand autologous groups. A total of eleven bacterial groups weresignificantly increased (1.5-2.5 fold) upon allogenic microbial fecalinfusion and contributed significantly to the separation of the groups.These include those related to the well-known butyrate-producerRoseburia intestinalis, the oxalate-converting Oxalobacter formigenes,various Ruminococci and other Firmicutes.

Effect of Fecal Transplant on Gut Microbiota in the Small Intestine

Total numbers of small intestinal bacteria did not change followingmicrobial fecal infusion. A set of seven bacteria significantlyassociated with the difference in biopsies of the small intestinebetween the allogenic and autologous groups was detected at six weeks(Table 1). In additional analyses, a significant association (r=0.8,p<0.01) was found between small intestinal Eubacterium halliiconcentrations and the improvement in insulin sensitivity (Rd) in humansubjects with metabolic syndrome six weeks after lean donor fecaltransplantation. Additionally, a significant correlation was found(r=0.6, p<0.05) between small intestinal Alcaligenes faecalisconcentrations and the improvement in insulin sensitivity (Rd) in humansubjects with metabolic syndrome six weeks after lean donor fecaltransplantation. Notably, E. hallii was almost two-fold reducedfollowing infusion in the autologous group. Other bacteria that werespecifically increased in the autologous group in comparison with theallogenic group include ileum-inhabitants such as Lachnobacillus bovis,Streptococcus bovis, and Prevotella ruminicola. Corynebacterium spp.were reduced in the allogenic but increased in the autologous group.Finally, bacteria related to the Gram-negative Escherichia coli showedan almost two-fold decrease in the allogenic group and a two-foldincrease in the autologous group (Table 1).

TABLE 1 Change in jejunal mucosal microbiota following allogenic fecaltransplant (n = 9 per group). Allogenic Group Autologous GroupFold-change Fold-change after/before after/before Phylum level BacterialGroup transplantation transplantation Firmicutes Eubacterium hallii etrel. 1.09 0.61 Proteobacteria Alcaligenes faecalis et rel. 1.18 0.97Firmicutes Streptococcus bovis et rel. 0.89 1.23 FirmicutesLachnobacillus bovis et rel. 0.63 0.98 Actinobacteria Corynebacteriumspp. 0.87 1.34 Proteobacteria Escherichia coli et rel. 0.58 2.21Bacteroidetes Prevotella ruminicola et rel. 0.99 1.01

Example 2

Eubacterium hallii L2-7 as described by Barcenilla et al. (2000, Appl.Environ. Microbiol., April, 66(4):1654-61; DSM 17630; obtained from thelaboratory of Prof. Harry Flint, Rowett Research Institute, Aberdeen,Scotland, UK) was grown in two bottles of 500 ml of Wilkins-Chalgrenmedium (1976, Antimicrob. Agents Chemother. 10:926-928) under anaerobicconditions till approximately 2×10⁹ cells per ml. Subsequently, thecultures were centrifuged (10,000 rpm in 15 minutes at 4° C.), washedtwice with anaerobic PBS (20 mM, pH 7, as detailed on the World Wide Webat en.wikipedia.org/wiki/Phosphate_buffered_saline) and re-suspended in20 ml of 10% glycerol in 20 mM PBS with 20 mM glucose and 20 mMmaltodextrin and frozen at −80° C. in aliquots of 100 μl containingapproximately 10¹¹ cells per ml. All manipulations were performed underanaerobic conditions.

Example 3

Eight-week-old db/db male mice on a C57BL6 background as well as maleC57BL6 mice were acquired from Jackson Laboratory (Bar Harbor, Me., USA)and allowed to acclimatize at the AMC animal facility (ARIA) during twoweeks before starting experiments. Mice were in a constant 12-hourlight-dark cycle with controlled temperature and humidity and were givenaccess to food (regular chow diet) and water ad libitum. Bodyweight wasmeasured once a week. Starting at the age of ten weeks, E. hallii wasorally administered at 10⁶, 10⁸ or 10¹⁰ CFU in 100 μl high glucosevehicle (20 mM). The solution was administered by daily oral gavage inthe morning using a 21-gauge syringe for 14 days (n=8 per group).Administration of only vehicle served as control. The cultured E. halliiwas administered orally to db/db mice (n=8 per group) for two weeks inincreasing doses (10×E10/100 μl, 10×E8/100 μl and 10×E6/100 μl ordissolvens (saline+glycerol), respectively). Their effect on lipidprofiles (measurement of total cholesterol, LDLc, HDLc and TG in fastingplasma samples as described in Example 1), fasting plasma glucose andinsulin levels for insulin resistance (HOMA), as well as postprandialglucose (oral glucose tolerance test), are determined as described abovein Example 1. Levels of short-chain fatty acids acetate, butyrate andpropionate are determined in peripheral and portal blood by MassSpectrometry (see Vrieze et al., Gastroenterology Jun. 20, 2012, Epubahead of print). Moreover, after sacrificing the mice, small intestinaland fecal samples are studied for E. hallii concentrations.

In this experiment, we find distinct effects of short-term oral E.hallii L2-7 supplementation to the small intestine on normalization ofinsulin resistance (as detected by HOMA calculation and postprandialglucose metabolism by AUC of oral glucose tolerance curve), as well asfasting lipid profiles in db/db mice.

1. A method of preventing and/or treating a subject suffering frominsulin resistance and/or insulin resistance-related complications, themethod comprising: administering to the subject Eubacterium hallii etrelatives and/or Alcaligenes faecalis et relatives so as to prevent ortreat insulin resistance and/or insulin resistance-relatedcomplications.
 2. The method according to claim 1, wherein the insulinresistance-related complications are selected from the group consistingof metabolic syndrome, dyslipidemia, and type 2 diabetes mellitus. 3.The method according to claim 1, wherein the Eubacterium hallii etrelatives and/or Alcaligenes faecalis et relatives is administered as apharmaceutical, food, or feed composition comprising the E. hallii etrel. and/or A. faecalis et rel.
 4. The method according to claim 3,wherein the insulin resistance-related conditions are selected from thegroup consisting of metabolic syndrome, dyslipidemia, and type 2diabetes mellitus.
 5. A method for preventing and/or treating insulinresistance and/or insulin resistance-related complications in a subjectin need thereof, the method comprising: increasing the level ofEubacterium hallii et relatives and/or Alcaligenes faecalis et relativesin the subject's small intestine.
 6. The method according to claim 5,wherein the level of Eubacterium hallii et relatives and/or Alcaligenesfaecalis et relatives in the subject's small intestine is increased by amethod selected from the group consisting of administering an effectiveamount of Eubacterium hallii et relatives and/or Alcaligenes faecalis etrelatives to the subject, and administering an effective amount of acompound capable of increasing the level of Eubacterium hallii etrelatives and/or Alcaligenes faecalis et relatives in the smallintestine.
 7. The method according to claim 5, wherein the insulinresistance-related conditions are selected from the group consisting ofmetabolic syndrome, dyslipidemia, and type 2 diabetes mellitus. 8.Pharmaceutical, food, or feed composition comprising Alcaligenesfaecalis et relatives.
 9. Composition according to claim 8, which is adrink.
 10. A method of reducing an insulin resistance-relatedcomplication in a subject diagnosed as suffering therefrom, the methodcomprising: administering to the subject an amount of a pharmaceutical,food, or feed composition comprising Alcaligenes faecalis et relativeseffective to reduce the insulin resistance-related complication.
 11. Themethod according to claim 6, wherein the insulin resistance-relatedconditions are selected from the group consisting of metabolic syndrome,dyslipidemia, and type 2 diabetes mellitus.
 12. A method of reducing aninsulin resistance-related complication in an obese subject diagnosed assuffering from the insulin resistance-related complication, the methodcomprising: administering to the subject an amount of Eubacterium halliiet relatives and Alcaligenes faecalis et relatives effective to reducethe insulin resistance-related complication.
 13. The method according toclaim 12, wherein the insulin resistance-related complication isselected from the group consisting of metabolic syndrome, dyslipidemia,type 2 diabetes mellitus, and combinations of any thereof.
 14. Themethod according to claim 12, wherein the E. hallii et rel. and/or A.faecalis et rel. is administered as a composition comprising both the E.hallii et rel. and A. faecalis et rel.
 15. The method according to claim14, wherein the insulin resistance-related condition is selected fromthe group consisting of metabolic syndrome, dyslipidemia, type 2diabetes mellitus, and combinations of any thereof.